The present invention relates to plants of the genus Raphanus that contain increased levels of anthocyanins, In particular the invention relates to edible Raphanus sprouts containing increased levels of anthocyanins, as well as to methods for their production.
There is an increasing consumer demand for edible young vegetable plants, also referred to as sprouts. Sprouts may be produced by germinating seeds in either liquid medium and harvested before the cotyledons appear, as is e.g. done with alfalfa. Alternatively, seeds may be sown onto a water-soaked solid support, such as cellulose, and allowed to germinate and grow in e.g. small disposable containers until or beyond the cotyledons appear. Further growth of the plantlets may be arrested, e.g. by cooling the plantlets, usually before the plants reach a height of about 4 to 15 cm. The plantlets are then ready for consumption. Sprouts of e.g. cress, daikon (a type of radish sprout) and mustard grow this way are very popular and many other vegetables and herbs are nowadays also available in the form of sprouts. The popularity of vegetable sprouts may at least in part be explained by the notion that they are healthy. Sprouts are indeed known to be very rich in vitamins and minerals such as the vitamins C and B1, potassium, calcium, phosphorus, magnesium and iron, while at the same time they are low in calories. Daikon sprouts, i.e. Sprouts of Raphanus sativa longipinatus, are particularly popular in the U.S, and in Japan were they are usually sold as young two-leafed green plants. Unfortunately, daikon sprouts do not contain anthocyanins, a compound that could further contribute to the health promoting effect of these sprouts.
Anthocyanins are present in many plants of higher order where they are responsible for the red, violet, blue or bluish black colours of flowers and fits. They are heterocyclic 2-phenyl-chromenol multiring systems (see also Formula 1) of varying hydroxylation patterns and varying absorption spectra in the visible light range. The sugar-free aglycon components of anthocyanins are referred to as anthocyanidins. They are obtained easily by hydrolysis of the glycosides contained in common fruits.
More recently, anthocyanins have drawn attention for their health promoting effects (see e.g. WO 92/03146). E.g, it is known that anthocyanins can act as scavengers for oxygen radicals such as superoxide anion radical, hydrogen peroxide, hydroxyl radical, alkoxyl radicals, peroxyl radicals for singulett oxygen, and many other radicals. Anthocyanins have also been described as photobiological inhibitors that intervene as regulators and detoxifiers in sensitised photoreactions which take place through oxygen, thereby preventing the radical and radical chain reactions which damage cells and nucleic acids and protein molecules. Anthocyanins also protect against cell toxic and carcinogenic aldehydes such as e.g. 4-hydroxy-hexenal, 4-hydroxy-octenal, 4-hydroxy-nonenal, propanal, butanal, pentanal, hexanal, 2,4-hepta-dienal, malonic dialdehyde, and others. They even prevent the formation thereof within the framework of lipoperoxidative chain reactions. As such they may aid in the prevention of cancer or may delay the effects of ageing. Furthermore, they detoxify the acetaldehyde resulting from ethanol decomposition and the formaldehyde resulting from methanol decomposition.
Some species of Raphanus do produce anthocyanins, as is most notable from the red colour on the outside of the radish varieties as usually sold in Europe and the U.S. However, no Raphanus sprouts are available that contain appreciable levels of anthocyanins. Thus, it is an object of the present invention to provide for Raphanus plants containing increased levels of anthocyanins, in particular, it is an object of the present invention to provide for Raphanus plants, the sprouts of which contain increased levels of anthocyanins. Advantages of the anthocyanin containing sprouts of the invention over other consumable anthocyanin sources, such as e.g. fruits like blueberry or grapes, include (1) the much shorter cultivation time; (2) the relatively high concentration of anthocyanins in the sprouts allowing to consume only small amounts of the sprouts to meet a certain anthocyanin intake; and (3) the great variety a dishes and recipes in which the sprouts may be applied.
In a first aspect the invention relates to a plant of the genus Raphanus, whereby the plant upon germination of its seed produces a sprout that comprises one or more anthocyanins at a level of at least 100 nmol per gram fresh weight of sprout. A Raphanus sprout is herein defined as any developmental stage of a Raphanus plant ranging from a germinating seed to a plantlet that has a height of no more than 20, preferably no more than 17, 15, 14, 12 or 10 cm. Preferably, a Raphanus sprout is a Raphanus plant in a developmental stage beyond a germinating seed and preferably having no more than two leaves, i.e. the cotyledons. Further preferred embodiments of Raphanus sprout are herein defined below.
The Raphanus plants of the invention, or sprouts or turnips thereof, comprise anthocyanins at a level of preferably at least 100, 200, 400, 800, 1500, 3000, 4000, 5000 or 6000 nmol per gram of fresh weight plant material. Fresh plant material of the Raphanus plants of the invention has an anthocyanin content of preferably at least 100, 200, 400, 800, 1500, 2500, 3500 or 4500 ppm (parts per million). The anthocyanin levels or content may be determined photospectrometrically, using a malvine calibration curve to estimate the anthocyanin levels or contents of anthocyanins extracted from fresh plant material as described in Example 4. Preferably, these anthocyanin levels are present in sprouts of the Raphanus plants of the invention. It is to be understood that these anthocyanin levels relate to the total level of anthocyanins and thus may comprise the various glycosylation forms of the anthocyanins as well as anthocyanins with different anthocyanidin moieties as defined below. The anthocyanin levels are expressed per gram fresh weight of plant material, whereby preferably, if present, the roots have been removed by cutting prior to the determination of the fresh weight of the material to be extracted. Alternatively, the anthocyanin levels in the Raphanus plants of the invention may be defined by comparison to a reference plant of the invention such as the Raphanus sativa line V33, (i.e. ATCC No. PTA-3630). Thus, a Raphanus plants of the invention preferably has an anthocyanin content of preferably at least 2, 5, 10, 20, 50 or 75% of the anthocyanin content of the Raphanus sativa line V33, (i.e. ATCC No. PTA-3630), whereby tie anthocyanin content of both plants is determined in the same part(s) of the plants and/or at the same developmental stage of the plants and using tie same analytical technique.
The Raphanus plants of the invention comprise anthocyanins. Anthocyanin are herein defined as compounds having the following characteristics, (1) comprising a molecular structure as shown in Formula 1, having a 2-phenyl benzopyrylium cation (flavylium ion); (2) an intense red, pink, violet, or purple colour; (3) a strong shift in colour at high alkaline pH (towards green to yellow); (4) solubility in water. The anthocyanins contained in the Raphanus plants of the invention preferably comprise an anthocyanin having a anthocyanidin moiety with the structure of Formula 1, wherein R1 is OH or OCH3, and wherein R2 is H, OH, or OCH3. The anthocyanin may contain one or more glycosides attached to any of the hydroxyl groups of the anthocyanidin moiety, but not to each of the hydroxyl groups, whereby preferably at least the hydroxyl group in the 3 position is glycosylated. The Raphanus plants of the invention comprise anthocyanins that preferably have an absorbance maximum at a wavelength higher than 515, 520, 525, or 530 nm and preferably at wavelength less than 550, 545, 540 or 535 nm. The Raphanus plants of the invention preferably comprise anthocyanins that comprise an anthocyanin having an anthocyanidin moiety selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin and malvidin, and more preferably the anthocyanins comprise an anthocyanin wherein the anthocyanidin moiety is malvidin.
The Raphanus plant of the invention preferably is a plant of the species Raphanus sativa, more preferably the plant is obtained through breeding and selection from the Raphanus sativa lines CGN 6924, CGN 7240, or both. Most preferably, the Raphanus plants of the invention are obtained through breeding and selection from the Raphanus sativa line V33, (i.e. ATCC No. PTA-3630).
Methods for breeding and selection of the Raphanus plants of the invention are well known to the skilled person. Generally such methods may include self- and cross-pollination and selection of new lines on the basis of colour, colour intensity, time of fading of (purple) colour to green, seed production, uniformity of germination time, height/length of the plant(let)s, absence of green descendants, taste, and size/shape of cotyledons. In order to intensify breeding, two generations may be grown per year, alternating growth in the Northern and Southern Hemisphere. In case interesting lines have not produced (sufficient) seed it may be necessary to maintain the line vegetatively in order to re-attempt seed production in the next season, This may require cutting of plants fully flowering. Several methods for cutting such plants are known in the art. A particularly advantageous method concerns cutting small shoots on the radish turnip together with some turnip tissue, treating it with cuttings-powder and incubating the slip under high humidity to grow roots.
In another aspect the invention relates to a Raphanus plant of the invention, wherein the plant is a sprout as herein defined above. In a preferred embodiment of the invention the sprout is a sprout prior to the two-leafed stage, i.e, so-called xe2x80x9calfalfa-type sproutsxe2x80x9d. Such sprouts may be cultured by methods and packaged in suitable containers for shipping and marketing as herein described below, The containers preferably contain a plurality of such sprouts wherein the sprouts are preferably ready for consumption.
In yet another aspect the invention relates to a Raphanus plant of the invention, wherein the plant is a plantlet that has at least two leaves. Preferably the plantlet has two leaves or no more than two leaves. The height or length of the plantlets, as measured from the root-tip to the top of the plantlet, is preferably at least 3, 4, or 5 cm and is preferably less than 20, 17, 15, 14, 12, 10, 8 or 6 cm. These plantlets/sprouts may be referred to as xe2x80x9ccress-type sproutsxe2x80x9d and are generally also referred to as micro-vegetables in the art. Such sprouts may be cultured by methods and packaged in suitable containers for shipping and marketing as herein described below. The containers preferably contain a plurality of such sprouts wherein the sprouts are preferably ready for consumption. Preferably the container contains at least 1, 2, 3, 4, 5, 6, or 8 plantlets per cm2.
In a further aspect the invention relates to material from a Raphanus plant of the invention as herein defined above. The material may be a root, a stem, a stalk, a leaf, a petal, a siliqua, a seed, a turnip, pollen, meristem, callus, a sepal, a flower, a cell, tissue or a combination thereof. The material may suitably be used for various purposes, including breeding or culturing the Raphanus plants of the invention, as food or a food ingredient, as (part of) a pharmaceutical composition or for isolation of anthocyanins that may be used for various purposes as herein defined below.
The turnips of the Raphanus plants of the invention are characterised in that the anthocyanins are not (only) present in the skin but also in the turnip itself, which is in contrast to the usual red radish that is has a white core and where the anthocyanins are only present in its skin. Thus, in a further aspect the invention relates to a Raphanus plant that produces turnips containing anthocyanins within the tip itself, i.e. in the core of turnip and/or throughout the turnip. Preferably, the turnip may contain anthocyanins with an absorbance maximum at a wavelength higher than 500 or 505 nm and less than 520 or 515 nm or around 510 nm. The turnip may preferably contain anthocyanins with an absorbance maximum at a wavelength higher than 510 or 515 nm and less than 540 or 535 nm, or around between 520 and 530 nm. The anthocyanin content of the up is preferably as herein defined above.
In a further aspect the invention rein to methods for cultivating and/or producing the purple Raphanus sprouts of the invention. Preferably, such methods produce sprouts suitable for human (or animal) consumption, i.e. the sprouts are edible. A sprout is suitable for human consumption if it does not have non-edible substrate such as soil attached or clinging to it, Typically the sprouts are grown on a non-nutritive solid support, such as agar, paper towel, blotting paper, Vermiculite, Perlite, etc., with water and light supplied. A particularly preferred non-nutritive solid support for use in the method of the invention is cellulose which may be applied in the form of pads, sheets or particles such as e.g. described in Dutch Patent No. 1001570. Thus, if a sprout is not grown in soil, but on a solid support there is no need for it to be washed to remove non-edible soil. If a sprout is grown in a particulate solid support, such as soil, Vermiculite, Perlite, or cellulose, washing may be required to achieve a sprout suitable for human consumption.
The Raphanus sprouts of the invention are preferably grown in containers which are suitable for shipping and marketing, Typically such containers are plastic boxes or jars which contain a wetted pad at the bottom. The containers allow light to penetrate while providing a mechanically protective barrier. Numerous methods for the cultivation of sprouts are known, as exemplified by Dutch Patent No. 192969; U.S. Pat. Nos. 3,733,745; 3,643,376; 3,945,148; 4,130,964; 4,292,760 or 4,086,725; Steve Meyerowitz, In: xe2x80x9cThe Complete Guide to Sprouting, Sprouts The Miracle Foodxe2x80x9d Sproutman Publications, May 1998; James C. Schmidt, Horticulture Facts, xe2x80x9cGrowing Sprouts Indoorsxe2x80x9d, (Rev. 4/81). The Raphanus sprouts of the instant invention can be stored and shipped in diverse types of containers such as jars, bags and boxes, open or closed, i.e. with or without lids, among many others. Any of the known methods for growing, packaging and shipping of sprouts are suitable.
Methods for producing Raphanus sprouts of the invention at least comprise the steps of (a) germinating seed of a Raphanus plant as herein defined above, in a suitable medium, under suitable conditions, and optionally in a container; and (b) growing the germinated seeds obtained in (a) under suitable conditions until a sprout of a desired developmental stage is obtained. The desired developmental stage may be a sprout prior to the appearance of the cotyledons, or may be a plantlet having at least two leaves and preferably no more than two leaves. A suitable growth medium preferably just contains water. Depending on the local quality of the tap-water further purification of the water may be required to demineralise the water and/or to remove chlorine, organic residues or other contaminants. The water is preferably free of microbial contaminants. Germination is preferably cared out at a temperature of 15-25xc2x0 C., preferably at high humidity, most preferably a relative humidity of 100%. Germination may suitably be performed in a germination cell or room with controlled temperature and humidity.
A preferred embodiment of the invention concerns a method for producing Raphanus sprouts of the Alfalfa-type. The methods applied are essentially similar to those used for producing Alfalfa sprouts. Seeds are washed and/or soaked and placed in a rotating drum or container. Preferably this is done under climate controlled conditions with optimal (near 100%) humidity. The seeds rotate fast enough to avoid clinging of the seeds, yet slow enough to avoid damage to the germs. Germination may be performed with or without light and preferably at a temperature between 15 and 25xc2x0 C. The temperature may be varied during the growth period. When the sprouts have grown to a desired stage, usually after about 100 hours (plus or minus 48 hours), they are harvested from the drum or container and may be washed with water. The sprouts may then be packaged into a variety of containers such as jars, bags and boxes in unit quantities ranging from 25 grams to bulk packing.
A further preferred embodiment of the invention concerns a method for producing Raphanus sprouts in the form of plantlets having at least two leaves and preferably no more than two leaves, i.e. preferably only having cotyledons and no true leaves. The method comprises the steps of (a) germinating seed of a Raphanus plant as defined herein above on a non-nutrative solid support (as described herein above) containing water and preferably no fertiliser or other additions, at a density of 3-12 seeds per cm2, at a temperature of 10-35xc2x0 C., preferably 15-25xc2x0 C., at high humidity and, optionally in the dark; (b) growing the germinated seeds obtained in (a) at a temperature of at least 10xc2x0 C. but no more than 35xc2x0 C., preferably 15-25xc2x0 C., at a humidity of at least 70% and under a daily cycle of light, until the germinated seeds have grown into plantlets having at least two leaves and a height of preferably at least 3, 4, or 5 cm; and optionally, (c) arresting further growth of the plantlets by cooling to a temperature between 1 and 6xc2x0 C., more preferably the plantlets are cooled to a temperature between 1.5 and 4xc2x0 C., most preferably to a temperature of about 2xc2x0 C.
A further aspect of the invention relates to the (mass)production of seed for the purple Raphanus sprouts. Seed of a suitable Raphanus line producing purple sprout is sown in a field with suitable soil and a suitable climate, Seed is preferably sown in the months of October to May in the Northern Hemisphere or in the months of April to September in the Southern Hemisphere. Plants are grown and allowed to flower and pollinated with the help of insects, preferably bees. The plants are allowed grow further and to ripen with seeds. The plants are then preferably mown in a swath, after which they continue to ripen and are allowed to dry under the influence of wind and sun. A threshing machine is preferably used to harvest the seeds, which may be further selected on the basis of colour, shape and size, such that uniform batches of seed are obtained.
In a further aspect the invention relates to methods for producing anthocyanin, wherein the anthocyanin preferably is an anthocyanin as herein defined above. The method comprises the steps of (a) growing a Raphanus plant of the invention; (b) harvesting the Raphanus plant or a part thereof; (c) recovery of the anthocyanins in the plant or part thereof; and (d) optionally purification of the anthocyanins. The Raphanus plant to be applied in the method may be any Raphanus plant as herein defined. The entire Raphanus plant may be harvested, which may a sprout or a plantlet as herein defined above. Alternatively, anthocyanin-rich parts of the plants may be harvested such as e.g., the leaves or turnips of the plants. Recovery of the anthocyanin will usually comprise some form of grinding or homogenisation of the plant or parts thereof and may farther comprise extraction of the anthocyanins with water or preferably a diluted acid in water, such as e.g. 5% formic acid, or with organic solvents such as methanol, which may preferably also contain an acid, such as e.g. 5% formic acid. Methods for recovery and extraction of anthocyanins from plant materials are well known in the art and may likewise be applied to the Raphanus plants of the invention. The anthocyanins may be flier purified by e.g. chromatography as described in the Examples below (see also Fiorini, 1995, J. Chromatogr, 692: 213-219). The thus obtained anthocyanin preparations may be used for a variety of purposes, including e.g. their incorporation into foods or drinks, as a food ingredient, as a natural colouring agent or dye for food, drinks or other materials, as a (food or pharmaceutical grade) preservative, in particular for the preservation of oxidation sensitive compositions or they may be formulated in to food supplements or into pharmaceutical compositions in a manner known per se for anthocyanins from other sources, as e.g. described in WO 92/03146. Such pharmaceutical compositions may be used in the treatment of wounds, ulcers, inflammatory symptoms, and pathogenic conditions of the vascular system or of disturbances caused by a deterioration of the lipoid or glycide metabolisms. In addition, compositions comprising the anthocyanins of the invention may be used to improve (night) vision and/or treat tired eyes. Such compositions may further be used in the prevention of cancer and cardiovascular disease, and in reducing the effects of ageing, such as impaired memory. Likewise, the anthocyanin containing plants of the invention, or past thereof may be used as such for the above purposes.
The advantages of the invention include the plants, plantlets, sprouts and plant parts of the invention possessing health promoting effects as compared to the prior art Raphanus plants. These plants or parts thereof may thus be used as nutraceuticals or functional foods. A further advantage of these plants and part thereof is that they have an attractive colour that may be used to add a new decorative aspect to recipes and dishes. The plants of the invention may further advantageously be applied as source for anthocyanins, which may be isolated therefrom for a variety of purposes including active ingredients in pharmaceutical compositions, food ingredient and/or as natural dye. Finally, the plants of the invention have been obtained through classical breeding techniques from naturally occurring isolates. As such these plants are non-GMO, thereby greatly increasing the public acceptance of these plants or products therefrom.